Chemical and biological separations are routinely performed in various industrial and academic settings. For example, recent developments in the pharmaceutical industry and in combinatorial chemistry have exponentially increased the number of potentially useful compounds, each of which must be characterized in order to identify their active components and/or establish processes for their synthesis. To more quickly analyze these compounds, researchers have sought to automate analytical processes and to implement analytical processes in parallel.
One useful analytical process is chromatography, which encompasses a number of methods that are used for separating ions or molecules that are dissolved in or otherwise mixed into a solvent. In fact, chromatography has many applications including separation, identification, purification, and quantification of compounds within various mixtures.
Liquid chromatography is a physical method of separation wherein a liquid “mobile phase” (typically consisting of one or more solvents) carries a sample containing multiple constituents or species through a separation medium or “stationary phase.” Various types of mobile phases and stationary phases may be used. Stationary phase material typically includes a liquid-permeable medium such as packed granules (particulate material) disposed within a tube (or other channel boundary). The packed material contained by the tube or similar boundary is commonly referred to as a “separation column.” High pressure is often used to obtain a close-packed column with a minimal void between each particle, since better resolution during use is typically obtained from more tightly packed columns. As an alternative to packed particulate material, a porous monolith may be used.
Typical interactions between stationary phases and solutes include adsorption, ion-exchange, partitioning, and size exclusion. Typical types of stationary phases to support such interactions are solids, ionic groups on a resin, liquids on an inert solid support, and porous or semi-porous inert particles, respectively. Commonly employed base materials include silica, alumina, zirconium, or polymeric materials. A stationary phase material may act as a sieve to perform simple size exclusion chromatography, or the stationary phase may include functional groups (e.g., chemical groups) to perform other (e.g., adsorption or ion exchange separation) techniques.
Mobile phase is forced through the stationary phase using means such as, for example, one or more pumps, gravity, voltage-driven electrokinetic flow, or other established means for generating a pressure differential. After sample is injected into the mobile phase, such as with a conventional loop valve, components of the sample will migrate according to interactions with the stationary phase and the flow of such components are retarded to varying degrees. Individual sample components may reside for some time in the stationary phase (where their velocity is essentially zero) until conditions (e.g., a change in solvent concentration) permit a component to emerge from the column with the mobile phase. In other words, as the sample travels through voids or pores in the stationary phase, the sample may be separated into its constituent species due to the attraction of the species to the stationary phase. The time a particular constituent spends in the stationary phase relative to the fraction of time it spends in the mobile phase will determine its velocity through the column. Following chromatographic separation in the column, the resulting eluate stream (consisting of mobile phase and sample) contains a series of regions having elevated concentrations of individual species, which can be detected by various techniques to identify and/or quantify the species.
As illustrated in FIG. 1, a separation column for use in a conventional hydrostatic pressure-driven chromatography system is typically fabricated by packing particulate material 14 into a tubular column body 12. A conventional column body 12 has a high precision internal bore 13 and may be manufactured with stainless steel, although materials such as glass, fused silica, and/or PEEK are also used. Various methods for packing a column body are known, although such methods are notoriously slow and laborious. In one example, a simple packing method involves dry-packing an empty tube by shaking particles downward with the aid of vibration from a sonicator bath or an engraving tool. A cut-back pipette tip may be used as a particulate reservoir at the top (second end), and the tube to be packed is plugged with parafilm or a tube cap at the bottom (first end). Following dry packing, the plug is removed and the tube 10 is then secured at the first end with a ferrule 16A, a fine porous stainless steel fritted filter disc (or “frit”) 18, a male end fitting 20A, and a female nut 22A that engages the end fitting 20A. Corresponding connectors (namely, a ferrule 16B, a male end fitting 20B, and a female nut 22B) except for the frit 18 are engaged to the second end to secure the dry-packed tube 12. The contents 14 of the tube 12 may be further compressed by flowing pressurized solvent through the packing material 14 from the second end toward the first (frit-containing) end. When compacting of the particle bed has ceased and the fluid pressure has stabilized, there typically remains some portion of the tube 13 that does not contain densely packed particulate material. To eliminate the presence of a void in the column 10, the tube 13 is typically cut down to the bed surface (or a shorter desired length) to ensure that the resulting length of the entire tube 12 contains packed particulate 14, and any unpacked tube section is discarded. Thereafter, the column 10 is reassembled (i.e., with the ferrule 16B, male end fitting 20B, and female nut 22B affixed to the second end) before use.
In another packing method utilizing slurry, an empty tube is attached to a packing reservoir such as a Poros® Self-Pack® reservoir (PerSeptive Biosystems, Foster City, Calif.) before being filled with an appropriate amount of dilute slurry. The end of the reservoir column is then screwed on firmly before the tube is internally pressurized with a fluid using an appropriate device such as a pump. Pressures of several hundreds or even thousands of pounds per square inch (psi) may be applied to pack the tube with particulate packing material, with the ultimate pressure depending on the properties of the tubing and the ability to seal the apparatus from leakage. A packed tube may be cut following the packing step to remove any dead volume (i.e., where packing is incomplete or not present) or to yield multiple sections, followed by the addition of end fittings to the uncapped tube ends to permit subsequent interface with fluidic components.
A conventional pressure-driven liquid chromatography system utilizing a column 10 is illustrated in FIG. 2. The system 30 includes a solvent reservoir 32, at least one high pressure pump 34, a pulse damper 36, a sample injection valve 38, and a sample source 40 all located upstream of the column 10, and further includes a detector 42 and a waste reservoir 44 located downstream of the column 10. The high pressure pump(s) 34 pressurize mobile phase solvent from the reservoir 32. A pulse damper 36 serves to reduce pressure pulses caused by the pump(s) 34. The sample injection valve 38 is typically a rotary valve having an internal sample loop for injecting a predetermined volume of sample from the sample source 40 into the solvent stream. Downstream of the sample injection valve 38, the column 10 contains stationary phase material that aids in separating species of the sample. Downstream of the column 10 is a detector 42 for detecting the separated species, and a waste reservoir 44 for ultimately collecting the mobile phase and sample products. A backpressure regulator (not shown) may be disposed between the column 10 and the detector 42. Many components of the system 30 are precision manufactured, thus elevating the cost of a typical high performance liquid chromatography system 30 to approximately $20,000-$30,000 or more. Given the rising demand for chromatographic separations, once such a system 30 is purchased, it may be operated on a nearly continuous basis.
The system 30 generally permits one sample to be separated at a time in the column 10. Due to the cost of conventional tubular chromatography columns, they are often re-used for many (e.g., typically about one hundred or more) separations. Following one separation, the column 10 may be flushed with a pressurized solvent stream in an attempt to remove any sample components still contained in the stationary phase material 14. However, this time-consuming flushing or cleaning step does not always yield a completely clean column 10. This means that, after the first separation performed on a particular column, every subsequent separation may potentially include false results due to contaminants left behind on the column from a previous run. Eventually, columns become fouled to the point that they are no longer useful, at which point they are removed from service and replaced.
A known method for increasing separation throughput is to modify a conventional chromatography system to split the flow from a common source of mobile phase (typically one or two pumps) to several chromatography columns. Such a system 50 is illustrated in FIG. 3. Mobile phase solvent from a solvent reservoir 52 is pressurized by one or more common high pressure pumps 54, and pressure variations caused by the common pump(s) 54 are damped by a common pulse damper 56. Downstream of the pulse damper 56, the solvent flow is split among multiple columns 10A, 10B, 10N each having an injection valve 58A, 58B, 58N and sample source 60A, 60B, 60N. (Although FIG. 3 shows three columns 10A, 10B, 10N, it will be readily apparent that any number of columns 10A, 10B, 10N may be provided. For this reason, the designation “N” is used to represent the last column 10N, with the understanding that “N” represents a variable and could represent any desired number of columns. This convention is used throughout this document.) Downstream of each column 10A, 10B, 10N is a detector 62A, 62B, 62N and waste reservoir 64A, 64B, 64N.
Compared to the single column system 30 described in connection with FIG. 2, the multi-column system 50 permits significantly higher throughput, since several samples can be analyzed in parallel. Additionally, this increased throughput may be obtained at a lower cost per separation, since the cost of expensive solvent delivery components (particularly the high pressure pumps 54 and pulse damper 56) can be spread over multiple columns 10A-10N. That is, a parallel chromatography system 50 having common solvent delivery components and several (i.e., “N”) separation columns is substantially less expensive than a comparable (“N”) number of chromatography systems each having discrete solvent delivery components. Moreover, the use of common solvent delivery components for a group of separation columns is substantially more compact than providing such components for each column, thus saving valuable laboratory space.
Despite the potential advantages of a multi-column separation system 50 having common mobile phase delivery components, such a system 50 presents complicating issues compared to the use of single column systems (such as the system 30 illustrated in FIG. 2). One issue is evenly splitting or balancing the flow of mobile phase through each column of the multi-column system 50. It would be desirable to provide the same flow conditions to each column of the multi-column system 50, but this is difficult to ensure for a number of reasons. To begin with, it can be difficult to precisely match the inlet and outlet volumes to each column when tubing and conventional connectors are used since tube lengths and flow characteristics through different connectors often vary. More importantly, individual columns tend to exhibit different fluidic impedance characteristics that prevent a common input stream from being divided evenly between the various columns since columns are typically packed one at a time or in an independent manner. Thus, depending on the specific fluid dynamics of a given packing process, the impedance characteristics of different columns typically vary significantly. In a linked multi-column system such as shown in FIG. 3, these column-to-column variations in flow may be caused by imperfect operation of any flow splitter(s) disposed upstream of the multiple columns, as well as by slight differences in column packing density, column geometry, and interfaces with the columns (e.g., fittings or other interfaces with fluidic supply components). It can be difficult to provide the same flow characteristics to each individual column of a group of columns due to these variations in fluidic impedance, since fluid flow will be biased toward the column(s) and overall flow paths with the least fluidic impedance.
To further complicate matters, many commonly employed chromatographic techniques utilize a “gradient” solvent profile that changes with time as opposed to an “isocratic” solvent profile that remains constant. For example, reverse phase chromatographic techniques often use an organic solvent/water gradient elution in which the concentration of the two solvents is varied with time by independently controllable pumps. Typically, separations in reverse phase chromatography depend on the reversible adsorption/desorption of solute molecules with varying degrees of hydrophobicity to a hydrophobic stationary phase. Thus, in a multi-column system employing a common set of pumps for performing gradient elution, the presence of a changing solvent concentration exacerbates the difficulty of ensuring that identical mobile phase conditions (i.e., including flow rate and concentration) are provided to each column at the same time.
Although active flow control systems (e.g., flow sensors and regulating valves) might be employed to reduce column-to-column fluid flow variation, active flow control systems are mechanically complex and expensive, and may not be suitable for use in extremely low flow environments. Moreover, active control systems may be difficult to tune so as to avoid hysteresis problems.
Due to the difficulty of providing identical mobile phase conditions (including both flowrate and mobile phase composition) to each column in a multi-column system utilizing common mobile phase supply components, seemingly identical columns tend to perform differently. That is, if the same sample is provided to each column in such a system, individual species exhibit different retention times from one column to another. As a result, it can be difficult to compare analytical results obtained from different columns in the same system.
Linking multiple discrete columns to a common mobile phase source raises some system packaging concerns. If the system is operated in gradient mode, it would be desirable to link the common mobile phase source to the columns with low volume conduit system to facilitate more rapid separation (i.e., by reducing the delay between the time a new solvent composition is generated and the time that new solvent composition actually reaches the separation columns). Additionally, it would be desirable to link the columns to one or more downstream detectors with low volume conduits to reduce diffusive mixing or band broadening between separated species following separation in the columns. With conventional multi-column systems, however, it may be difficult to physically interconnect all of the various fluid delivery and detection components without fairly significant conduit volumes.
Another potential complication associated with conventional multi-column separation systems—particularly those using threaded end-fittings—is that it is laborious to change columns when they are spent, and that the entire system is incapacitated during a such a procedure. If possible, it would be desirable to reduce unproductive downtime of a parallel separation system.
A further concern associated with the use of conventional multi-column separation systems is their increased consumption of both samples and reagents, leading to increased waste disposal quantities and attendant expenses. It would be desirable to provide a system that could provide high separation throughput without excessive sample and reagent consumption.
In light of the foregoing, there exists a need for an improved high-throughput separation system and methods for fabricating the same.